Table 4.

Experimental studies investigating the role of NO in IVF.

Authors	Model	NO Donors Used	NOS Inhibitors Used	Effects
Kim et al., 2004 [49]	Mice (In vivo)	-L-NAME (0.5, 1, 5 and 10 mM)		-L-NAME inhibited the fertilization rate and the early embryonic development by treating sperm or oocytes. -Fertilization rate and early embryonic development were reduced when L-NAME or L-arginine was added to the culture media of embryos. -Microinjection of L-NAME into the fertilized embryos inhibited in a dose-dependent manner early embryonic development, but only by high concentrations of L-arginine.
Santana et al., 2014 [50]	Bovine (in vitro embryo culture)	-L-NAME (10 mM) -L-arginine (1 mM, 10 mM, or 50 mM)		-Supplementation with L-NAME from Day 1 to 8 of the culture decreased blastocyst and hatching rates. -L-arginine (50 mM) added from Day 1 to Day 8 decreased the blastocyst rates; in contrast, when added from Day 5 to 8, L-arginine (1 mM) improved the embryo hatching rates and quality. -Positive correlation between NO levels in the medium during this culture period and increased embryo hatching rates and quality.
Barroso et al., 1998 [51]	Mice (In vitro embryo culture and in vivo)	-DETA/NO: 0.001 mM, 0.01 mM, 0.1 mM, 1mM -DETA: 0.1 mM, 1 mM In-vivo: S.c. implantation of miniosmotic pumps containing either saline or different concentrations of DETA/NO or DETA (0.2, 0.4, and 0.8 mM) to deliver a daily dose of 5, 10 or 20 µmol per animal.		-None of the embryos progressed beyond the 4-cell stage when exposed to DETA/NO (0.1 or 1.0 mM). -Embryo development unaffected by lower (0.001 and 0.01 mM) concentrations of DETA/NO, after 48 h preincubation with DETA/NO or DETA only. -Embryo implantation inhibition with the infusion of DETA/NO in a dose-dependent manner. -No implantation sites observed with the infusion of a daily dose of 20 µmol DETA/NO, compared with the control or DETA-treated mice.
Tranguch et al., 2003 [52]	Mice (In vitro embryo culture)	-SNP: from 0.1 to 500 mM	-l-NA: from 125 to 500 mM	-All three NOS isoforms were expressed in two-cell, four-cell, morula, and blastocyst embryos. -Blastocyst-stage embryos isolated on the midmorning of Day 4 of pregnancy expressed only nNOS and eNOS, whereas those isolated in the midafternoon expressed all three NOS isoforms.
Gouge et al., 1998 [53]	Mice (in vitro embryo culture)		-l-NA: 500 µM	-Preimplantation murine embryos produced NO, reversibly inhibited by the culture of embryos in medium containing L-NA. -L-NA inhibits normal embryo development.
Chen et al., 2001 [54]	Mice (in vitro embryo culture)	-SNP: 0.1 µM, 1 µM, 10 µM	-L-NAME: 0.1 µM, 1 µM, 10 µM	-L-NAME inhibited blastocyst development in a concentration-dependent manner and SNP (0.1 µM) reversed this effect. -Excessive NO (> or = 10 µM) induced apoptosis in the mouse embryos. -The inhibitory effect of L-NAME was reversed by 8-Br-cGMP that rescued the embryo growth. -ODQ inhibited the embryo development in a dose-dependent manner (0.1 µM–100 µM) but had no effect on NO-induced embryo apoptosis.

8-Br-cGMP—8-Bromoguanosine 3′,5′-cyclic monophosphate; DETA/NO—Diethylenetriamine/nitric oxide; DETA—Diethylenetriamine; IVF—In-vitro fertilization; L-NAME—NG-nitro-l-arginine methyl ester; l-NA—NG-nitro-l-arginine; NO—Nitric oxide; NOS—Nitric oxide synthase; ODQ—1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; S.C—Subcutaneous; SNP—Sodium nitroprusside.